Abstract: Atherosclerosis is the direct cause of heart attack and stroke, the No. 1 and No. 3 killers in the United States, respectively. It is the result of both lipid deposition and chronic vascular inflammation. Toll-like receptors (TLRs), the key components of innate immunity, play detrimental roles in every stage of atherosclerosis, with TLR2 and TLR4 being best documented. While participating in the first line of defense against invading pathogens, TLR2- and TLR4-mediated signaling is considered to be a driving force in atherogenesis. Thus, blockade of TLR2 and TLR4 signaling is an intriguing therapeutic approach for atherosclerosis. However, no TLR2 or TLR4 antagonists are currently approved for clinic use. Recently, in an effort to isolate and characterize single compounds from Sparganium stoloniferum tubers, a commonly used Traditional Chinese Medicine herb, we obtained a novel compound, designated Sparstolonin B (SsnB), and made exciting discoveries. Our preliminary studies show that 1) SsnB has potent anti-inflammatory effects on macrophages by selectively blocking TLR2 and TLR4 signaling; 2) SsnB diminishes the ability of activated endothelial cells to attract monocytes for adhesion, and decreases arterial smooth muscle cell migration; and 3) SsnB effectively suppresses inflammatory response to lipopolysaccharide (LPS) in mice. On the basis of these preliminary data, we hypothesize that SsnB can be developed as an anti-inflammatory and anti-atherogenic agent by virtue of its selective inhibitory effects on TLR2 and TLR4 signaling. To test this hypothesis, we propose three specific aims. SA1. To elucidate the molecular mechanism by which SsnB blocks TLR2 and TLR4 signaling. We will express and purify the Toll/IL-1 receptor (TIR) domains of TLRs, the adaptor proteins TIRAP/Mal and MyD88, and examine the binding of SsnB to these proteins. SA2. To examine the effects of SsnB on resident vascular cells. We will test the hypothesis that SsnB suppresses the inflammatory phenotype in arterial endothelial and smooth muscle cells by blocking TLR2 and TLR4 signaling. SA3. To test the hypothesis that SsnB attenuates atherogenesis in mice. LDL receptor (LDLR) deficient mice will be fed high fat diet to induce hypercholesterolemia and atherosclerosis. SsnB will be administrated to test if it attenuates atherogenesis in these mice. In summary, this study will test the anti-inflammatory and anti-atherogenic effects of a new natural compound recently isolated and characterized by us. The confirmation of the hypothesis that SsnB has anti-inflammatory and anti-atherogenic merit by blocking TLR2 and TLR4 signaling in macrophages and resident vascular cells will usher the development of this compound into an anti-atherogenic agent. This study will also provide a new example of complementary and alternative medicine bridging with modern pharmacology.